This is a competing continuation proposal for a multi-investigator project to study and contravene EGFR signaling in cancer. The investigators bring to bear expertise in protein engineering (Wittrup, MIT), mass spectrometric phosphoproteomics (White, MIT), computational systems biology (Lauffenburger, MIT), and structural biology (Kuriyan, UC Berkeley). The first ten years of this project has produced 52 publications that have been cited in total 2,088 times, with a mean of 40 and a median of 24 citations per paper, and an h index of 26. In this renewal we propose to extend a novel therapeutic modality developed in the previous grant period, and to deepen our understanding of EGFR signaling networks and receptor structure/function relationships. We have created a novel triepitopic antibody topology by appending two small Fn3-based EGFR binding domains to the cetuximab IgG. This single construct binds at three nonoverlapping epitopes on EGFR, driving rapid clustering and downregulation without detectable receptor phosphorylation or downstream signaling. The triepitopic antibody controls growth of xenografted tumors that are resistant to the parent cetuximab antibody, indicating a qualitative improvement in mechanism of action that overcomes resistance due to KRAS and BRAF mutations. We will extend the functionality of this triepitopic construct to inhibit HER3 in order to pre-emptively overcome a key resistance mechanism. We will also employ a novel EGFR-targeted siRNA delivery vector to identify genes whose silencing produce antitumor efficacy synergistic with EGFR antagonism. We will apply our sophisticated experimental and computational network analysis tools to understanding three forms of resistance to anti-EGFR therapeutics: a) mutations in effector kinases such as KRAS and BRAF; b) upregulation of MET and HER3; and c) altered proteolytic shedding of ErbB ligands and ectodomains. In each case, signaling network interconnectivity and dynamics will be studied in untreated, cetuximab treated, and triepitopic antibody treated cell lines to examine how therapeutic interventions interact with the dysregulated pathways present in cancer cells. We will extend our structural studies of EGFR receptor biology to deepen our understanding of the flow of information from EGFR ligand binding outside the cell to kinase activation in the cytoplasm. Membrane protein NMR is revealing key conformational states of the EGFR transmembrane domain. Crystallographic, mass spectrometric, and fluorescence microscopic assays will help identify the temporal order and topological control of autophosphorylation in the activated EGFR dimer. This unusually comprehensively integrated multidisciplinary project has excellent momentum, and the team is enthusiastically engaged in pressing forward in these exciting new directions.